Immunization with live, attenuated vaccines has been the most effective strategy employed for the control of virus disease. However, undesirable complications can result from reversion of the attenuated vaccine strain to a strain which is virulent, in some cases inducing the disease syndrome that the vaccine was designed to prevent. The goal of the proposed research is to introduce multiple, well-defined attenuating mutations into the genome of the alphavirus, venezuelan eguine encephalitis (VEE), thus producing a live virus vaccine candidate strain wit an extremely low rate of reversion to virulence. First, we propose to identify the loci which control VEE virulence by sequencing a series of closely related attenuated and virulent VEE mutants which we have isolated previously. Second, the RNA genome of VEE will be converted to a form ammenable to genetic manipulation by construction of a full-length cDNA clone of VEE placed downstream from a T7 promoter. Infectious virus may be recovered from such cDNA clones by in vitro synthesis of infectious VEE RNA and transfection of cultured cells. Third, multiple, well- defined, attenuating mutations will be introduced into the full- length VEE construct by allelic replacement and/or site-directed mutagenesis. If each of these mutations independently renders the virus attenuated, then virus derived from such a construct will be much less likely to revert to virulence. Finally, the derived virus will be tested for pathogenicity, immunogenicity and reversion to virulence in animal models. We feel that successful completion of this study will make a significant contribution in four major areas. One, the availability of a full-length molecular clone of VEE will greatly facilitate the study of this important pathogen. Two, specific domains controlling virulence of VEE will be identified. Three, the potential usefulness of this approach in the design of live attenuated vaccines for positive-strand RNA viruses will be evaluated. And four, if the genetically engineered strains developed in this study prove superior to the existing investigational vaccine in animal models, they can be considered as candidates for use in humans.